JoVE Bioengineering
High-throughput Synthesis of Carbohydrates and Functionalization of Polyanhydride Nanoparticles
1Department of Chemical and Biological Engineering, Iowa State University, 2Department of Chemistry, Iowa State University
In this article, a high throughput method is presented for the synthesis of oligosaccharides and their attachment to the surface of polyanhydride nanoparticles for further use in targeting specific receptors on antigen presenting cells.
JoVE Application Notes
A New High Sensitivity Tandem Quadrupole Mass Spectrometer for Quantitative LC/MS/MS Analysis of Low Exposure Pharmaceuticals - ADVERTISEMENT
Pharmaceutical Business Operations, Waters Corporation
JoVE Neuroscience
Vertical T-maze Choice Assay for Arthropod Response to Odorants
Entomology and Nematology Department, University of Florida
A vertical, T-maze olfactometer is described for assaying the behavioral response of arthropods. The olfactometer allows the experimenter to measure choices performed by test subjects when subjected to two potential odor fields. Both attraction to and repulsion from odorants can be measured with this device.
JoVE General
Fabrication of the Thermoplastic Microfluidic Channels
Department of Biomedical Engineering, Boston University
Here we demonstrate how to fabricate thermoplastic microfluidic chips using hot embossing and heat sealing. Then we demonstrate how to use in situ light directed surface grafting and polymerization through the sealed chip to form the composite solid phase columns.
JoVE Bioengineering
Microfluidic Chip Fabrication and Method to Detect Influenza
1Department of Mechanical Engineering, Boston University, 2Department of Biomedical Engineering, Boston University
An integrated microfluidic thermoplastic chip has been developed for use as a molecular diagnostic. The chip performs nucleic acid extraction, reverse transcriptase, and PCR. Methods for fabricating and running the chip are described.
JoVE Bioengineering
A Toolkit to Enable Hydrocarbon Conversion in Aqueous Environments
1Department of Biotechnology, Delft University of Technology, 2Delft Center for Systems and Control, Delft University of Technology
A sustainable auto regulating bacterial system for the remediation of oil pollutions was designed using standard interchangeable DNA parts (BioBricks). An engineered E. coli strain was used to degrade alkanes via β-oxidation in toxic aqueous environments. The respective enzymes from different species showed alkane degradation activity. Additionally, an increased tolerance to n-hexane was achieved by introducing genes from alkane-tolerant bacteria.
JoVE General
Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans
1Center for Human Genetic Research and Department of Medicine, Massachusetts General Hospital and Harvard Medical School, 2Department of Earth, Atmospheric, and Planetary Sciences, Massachusetts Institute of Technology
We present robust biochemical and microscopic methods for studying Caenorhabditis elegans lipid stores. A rapid, simple, fixing-staining procedure for fluorescent lipid droplet imaging leverages the spectral properties of the lipophilic dye Nile red. We then present biochemical measurement of triglycerides and phospholipids using solid phase extraction and gas chromatography-mass spectrometry.
JoVE General
Electricity-Free, Sequential Nucleic Acid and Protein Isolation
1CUBRC, Inc., 2Department of Microbiology and Immunology, State University of New York at Buffalo, School of Medicine and Biomedical Sciences
A tool and chemistries are described to sequentially isolate nucleic acids followed by proteins from a sample without the need for electricity. The tool consists of a sorbent held within a transfer pipette while the isolation chemistries are based on solid-phase extraction principles. The isolated macromolecules can be analyzed by immuno-based and PCR-based assays.
JoVE Immunology and Infection
Quantification of Fungal Colonization, Sporogenesis, and Production of Mycotoxins Using Kernel Bioassays
Plant Pathology and Microbiology, Texas A&M University
The devastation of cereal crops by seed-infecting fungi has prompted numerous research efforts to better understand plant-pathogen interactions. To study seed-fungal interactions in a laboratory setting, we developed a robust method for the quantification of fungal reproduction, biomass, and mycotoxin contamination using kernel bioassays.
JoVE General
Solid Phase Synthesis of a Functionalized Bis-Peptide Using "Safety Catch" Methodology
College of Science and Technology, Temple University
The efficient solid-phase peptide synthesis of a functionalized bis-peptide trimer utilizing a "safety catch" cleavage procedure from HMBA resin is described.
JoVE General
Monitoring Plant Hormones During Stress Responses
Department of Biology, University of Texas
A simple method is provided that allows for the rapid extraction and analysis of multiple plant hormones from small tissue samples. The procedure uses vapor phase extraction as the solemn purification step. Samples are analyzed by GC/MS with chemical ionization that produces mainly (M+1)+ ions.
JoVE Immunology and Infection
Assessing Hepatic Metabolic Changes During Progressive Colonization of Germ-free Mouse by 1H NMR Spectroscopy
1School of Chemistry, Food and Pharmacy, The University of Reading, 2Department of Nutritional Sciences, The University of Reading
A progressive colonization procedure is described to further assess its impact on the host hepatic metabolism. Colonization is monitored non invasively by evaluating the urinary excretion of microbial co-metabolites by NMR-based metabolic profiling while hepatic metabolism is assessed by High Resolution Magic Angle Spinning (HR MAS) NMR profiling of intact biopsy.
JoVE Application Notes
BIOCIUS - RapidFire System - ADVERTISEMENT
RapidFire® technology enables label-free screening for lead discovery and in vitro ADME applications. The technology incorporates high-throughput solid phase extraction coupled to mass spectrometric analysis with analysis times of 6-8 seconds per sample. The system enables the direct measurement of historically difficult analytes without labels or surrogates. RapidFire technology is used by 13 of the top 15 pharmaceutical companies to accelerate their drug discovery efforts.
JoVE General
Purification of Transcripts and Metabolites from Drosophila Heads
1Department of Neurology, McKnight Brain Institute, University of Florida, 2Department of Entomology and Nematology, University of Florida, 3Genetics Institute, Department of Molecular Genetics and Microbiology, University of Florida, 4McKnight Brain Institute, Department of Neuroscience, Genetics Institute, Center for Translational Research on Neurodegenerative Diseases, and Center for Movement Disorders and Neurorestoration, University of Florida
We describe here the procedures for the extraction and purification of mRNA and metabolites from Drosophila heads. We are applying these techniques to better understand the cellular perturbations underlying neuronal degeneration. These methodologies can be easily scaled and adapted for other "omic" projects.
JoVE Applied Physics
Polycrystalline Silicon Thin-film Solar cells with Plasmonic-enhanced Light-trapping
School of Photovoltaics, University of New South Wales
Polycrystalline silicon thin-film solar cells on glass are fabricated by deposition of boron and phosphorous doped silicon layers followed by crystallisation, defect passivation and metallisation. Plasmonic light-trapping is introduced by forming Ag nanoparticles on the silicon cell surface capped with a diffused reflector resulting in ~45% photocurrent enhancement.
JoVE Applied Physics
Fabrication of Nano-engineered Transparent Conducting Oxides by Pulsed Laser Deposition
1Department of Energy and NEMAS - Center for NanoEngineered Materials and Surfaces, Politecnico di Milano, 2Center for Nano Science and Technology, Instituto Italiano di Tecnologia
We describe the experimental method to deposit nanostructured oxide thin films by nanosecond Pulsed Laser Deposition (PLD) in the presence of a background gas. By using this method Al-doped ZnO (AZO) films, from compact to hierarchically structured as nano-tree forests, can be deposited.
JoVE Immunology and Infection
Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce
1Center for Meat Safety and Quality, Department of Animal Sciences, Colorado State University, 2Rapid Microbial Detection and Control Laboratory, Department of Food Science and Human Nutrition, Iowa State University
This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).
JoVE General
The Logic, Experimental Steps, and Potential of Heterologous Natural Product Biosynthesis Featuring the Complex Antibiotic Erythromycin A Produced Through E. coli
1Chemical and Biological Engineering Department, State University of New York at Buffalo, 2Chemical Engineering Department, Massachusetts Institute of Technology
The heterologous biosynthesis of erythromycin A through E. coli includes the following experimental steps: 1) genetic transfer; 2) heterologous reconstitution; and 3) product analysis. Each step will be explained in the context of the motivation, potential, and challenges in producing therapeutic natural products using E. coli as a surrogate host.
JoVE Neuroscience
Microdialysis of Ethanol During Operant Ethanol Self-administration and Ethanol Determination by Gas Chromatography
College of Pharmacy, Division of Pharmacology and Toxicology, The University of Texas at Austin
A method to determine the time course of ethanol concentration in the brains of rats during operant ethanol self-administration is described. Gas chromatography with flame ionization detection is used to quantify ethanol in the dialysate samples, because it has the sensitivity required for the small volumes that are generated.
JoVE Bioengineering
Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis
1Centers for Cancer Pharmacology and Excellence in Environmental Toxicology, University of Pennsylvania, 2Department of Pharmacology, University of Pennsylvania
This protocol will demonstrate the extraction and analysis of free and esterified bioactive fatty acids from cells. Fatty acids are accurately quantified using stable isotope dilution, chiral liquid chromatography, electron capture atmospheric chemical ionization multiple reaction monitoring mass spectrometry (SID-LC-ECAPCI-MRM/MS).
JoVE General
Isolation and Biophysical Study of Fruit Cuticles
1Department of Chemistry, City College of New York, City University of New York Graduate Center and Institute for Macromolecular Assemblies, 2Department of Chemical Engineering, City College of New York
Aerial plant organs are protected by the cuticle, a supramolecular biopolyester-wax assembly. We present protocols to monitor selective removal of epi- and intracuticular waxes from tomato fruit cuticles on molecular and micro scales by solid-state NMR and atomic force microscopy, respectively, and to assess the cross-linking capacity of engineered cuticular biopolyesters.
JoVE Immunology and Infection
IgY Technology: Extraction of Chicken Antibodies from Egg Yolk by Polyethylene Glycol (PEG) Precipitation
1Center for Biological Security, Robert Koch-Institute, 2CICVyA - INTA Castelar, Instituto de Virología, 3Center of Molecular Immunology, Ciudad de la Habana, Cuba, 4Department of Biology, Chemistry, Pharmacy, Institute of Biology-Neurobiology, Free University of Berlin, 5Institut of Pharmacology, Charité-University Medicine of Berlin
This protocol describes in particular the extraction of total IgY from egg yolk by means of polyethylene glycol precipitation and gives general information about IgY technology.
JoVE Bioengineering
Solid-phase Submonomer Synthesis of Peptoid Polymers and their Self-Assembly into Highly-Ordered Nanosheets
Molecular Foundry, Lawrence Berkeley National Laboratory
A simple and general manual peptoid synthesis method involving basic equipment and commercially available reagents is outlined, enabling peptoids to be easily synthesized in most laboratories. The synthesis, purification and characterization of an amphiphilic peptoid 36mer is described, as well as its self-assembly into highly-ordered nanosheets.
JoVE Clinical and Translational Medicine
Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods
1Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran, 2Department of Endocrinology & Female Infertility, Reproductive Biomedicine Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran
The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.
JoVE Immunology and Infection
Preparation of Viral DNA from Nucleocapsids
Department of Molecular Biology, Princeton University
We describe the process of isolating high purity herpesvirus nucleocapsid DNA from infected cells. The final DNA captured from solution is of high concentration and purity, making it ideally suited for high-throughput sequencing, high fidelity PCR reactions, and transfections to produce new viral recombinants.
JoVE General
Maintaining Wolbachia in Cell-free Medium
Malaria Research Institute, Bloomberg School of Public Health, Johns Hopkins University
This video describes a method for purifying Wolbachia pipientis from an Anopheles gambiae cell line and then culturing the endosymbiont in cell-free medium. An assay for viability of the bacterium is demonstrated.
JoVE Chemistry
Large Scale Non-targeted Metabolomic Profiling of Serum by Ultra Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS)
Proteomics and Metabolomics Facility, Colorado State University
Non-targeted metabolite profiling by ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS) is a powerful technique to investigate metabolism. This article outlines a typical workflow utilized for non-targeted metabolite profiling of serum including sample organization and preparation, data acquisition, data analysis, quality control, and metabolite identification.
JoVE Clinical and Translational Medicine
Non-invasive Optical Measurement of Cerebral Metabolism and Hemodynamics in Infants
1Athinoula A. Martinos Center for Biomedical Imaging, Massachusetts General Hospital, Harvard Medical School, 2Lab. PALM, Université de Caen Basse-Normandie, 3Fetal-Neonatal Neuroimaging and Developmental Science Center, Boston Children's Hospital, Harvard Medical School, 4ISS, INC.
We combined frequency-domain near-infrared spectroscopy measures of cerebral hemoglobin oxygenation with diffuse correlation spectroscopy measures of cerebral blood flow index to estimate an index of oxygen metabolism. We tested the utility of this measure as a bedside screening tool to evaluate the health and development of the newborn brain.
JoVE General
Digital Microfluidics for Automated Proteomic Processing
1Department of Chemistry, University of Toronto, 2Donnelly Centre for Cellular and Biomolecular Research, 3Institute for Biomaterials and Biomedical Engineering, University of Toronto
Digital Microfluidics is a technique characterized by the manipulation of discrete droplets (~nL - mL) on an array of electrodes by the application of electrical fields. It is well-suited for carrying out rapid, sequential, miniaturized automated biochemical assays. Here, we report a platform capable of automating several proteomic processing steps.
JoVE General
Toxin Induction and Protein Extraction from Fusarium spp. Cultures for Proteomic Studies
Protein extraction for proteomic analyses in fungal species requires high levels of standardization to be accomplished according with the minimum information about a proteomic experiment (MIAPE) guidelines. We present a video-protocol that includes a procedure for minimizing experimental bias during toxin induction and protein extraction from Fusarium spp.
JoVE General
Electron Spin Resonance Micro-imaging of Live Species for Oxygen Mapping
Schulich Faculty of Chemistry, The Technion, Israel Institute of Technology
This protocol describes a method for micron-scale three-dimensional imaging of oxygen concentration in the immediate environment of live cells by electron spin resonance microscopy.
JoVE Applied Physics
Harvesting Solar Energy by Means of Charge-Separating Nanocrystals and Their Solids
1Department of Physics, Bowling Green State University, 2The Center for Photochemical Sciences, Bowling Green State University, 3Department of Chemistry, Bowling Green State University
A general strategy for the development of charge-separating semiconductor nanocrystal composites deployable for solar energy production is presented. We show that assembly of donor-acceptor nanocrystal domains in a single nanoparticle geometry gives rise to a photocatalytic function, while bulk-heterojunctions of donor-acceptor nanocrystal films can be used for photovoltaic energy conversion.
JoVE Clinical and Translational Medicine
Movement Retraining using Real-time Feedback of Performance
Department of Physical Therapy, University of British Columbia
Retraining abnormal movement patterns following injury or disease is a key component of physical rehabilitation. Recent advances in technology have permitted accurate assessment of movement during a variety of tasks, with near instantaneous quantification of results. This provides new opportunities for modification of faulty movement patterns in real time.
JoVE Application Notes
ヒト表皮再構築モデルを用いたIn vitro皮膚刺激試験 - ADVERTISEMENT
JoVE General
Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage (CoTrAC)
1Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, 2Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, 3Department of Physics, Jilin University
We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC), to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method has been used to follow the stochastic expression dynamics of a transcription factor, the λ repressor CI 1.
JoVE Neuroscience
In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons
1Unit on Neural Circuits and Adaptive Behaviors, Genes Cognition and Psychosis Program, National Institute of Mental Health, 2Department of Neuroscience, Brown University - National Institutes of Health Graduate Partnership Program, 3Section on Synaptic Pharmacology, Laboratory for Integrative Neuroscience, National Institute on Alcohol Abuse and Alcoholism, 4Champalimaud Neuroscience Programme, Champalimaud Center for the Unknown
Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.
JoVE General
Microfluidic Applications for Disposable Diagnostics
Department of Biomedical Engineering, Boston University
In this interview, Dr. Klapperich discusses the fabrication of thermoplastic microfluidic devices and their application for development of new diagnostics.
JoVE Bioengineering
Monitoring Protein Adsorption with Solid-state Nanopores
Department of Physics, Syracuse University
A method of using solid-state nanopores to monitor the non-specific adsorption of proteins onto an inorganic surface is described. The method employs the resistive-pulse principle, allowing for the adsorption to be probed in real-time and at the single-molecule level. Because the process of single protein adsorption is far from equilibrium, we propose the employment of parallel arrays of synthetic nanopores, enabling for the quantitative determination of the apparent first-order reaction rate constant of protein adsorption as well as and the Langmuir adsorption constant.
JoVE General
Counting Human Neural Stem Cells
Department of Pathology, University of California, Irvine (UCI)
Knowledge of the exact number of viable cells is required for many tissue culture manipulations. This protocol describes how to differentiate between live and dead cells and quantify cells using a hemacytometer. Although it describes counting human neural stem/precursor cells (hNSPCs), it can be used for other cell types.
JoVE General
DNA Extraction from Paraffin Embedded Material for Genetic and Epigenetic Analyses
1Department of Integrative Oncology, BC Cancer Research Centre, 2Interdisciplinary Oncology Program, University of British Columbia - UBC, 3Photography/Video Production, Multi-Media Services, BC Cancer Agency, 4Department of Pathology and Laboratory Medicine, University of British Columbia - UBC
This video demonstrates the protocol for DNA extraction from formalin-fixed paraffin-embedded material. This is a multi-day procedure in which tissue sections are deparaffinized with xylene, rehydrated with ethanol and treated with proteinase K to purify and isolate DNA for subsequent gene-specific or genome-wide analysis.
JoVE General
A Noninvasive Hair Sampling Technique to Obtain High Quality DNA from Elusive Small Mammals
We present a noninvasive sampling approach to efficiently collect hair samples from elusive small mammals, as shown for the American pika. We demonstrate the utility of this method by extracting DNA from sampled hair and amplifying several types of molecular markers commonly used in studies of wildlife ecology and conservation.
JoVE Bioengineering
Polymer Microarrays for High Throughput Discovery of Biomaterials
1Laboratory of Biophysics and Surface Analysis, University of Nottingham, 2School of Molecular Medical Sciences, University of Nottingham, 3David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology
A description of the formation of a polymer microarray using an on-chip photopolymerization technique. The high throughput surface characterization using atomic force microscopy, water contact angle measurements, X-ray photoelectron spectroscopy and time of flight secondary ion mass spectrometry and a cell attachment assay is also described.
JoVE General
Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture
Molecular Oncology Research Institute, Department of Medicine, Tufts Medical Center
A simple method is described for analyzing effects of tissue fibroblasts on associated epithelial cells. The combination of this method and three-dimensional tissue culture can facilitate analysis of cells after isolation from 3D. The technique is applicable to cells of varying malignant potential, allowing systematic study of effects of tumor-associated stroma on tumor cells.
JoVE Bioengineering
Cell Co-culture Patterning Using Aqueous Two-phase Systems
1Department of Biomedical Engineering, University of Michigan, 2Department of Macromolecular Science and Engineering, University of Michigan
Aqueous two-phase systems were used to simultaneously pattern multiple populations of cells. This fast and easy method for cell patterning takes advantage of the phase separation of aqueous solutions of dextran and polyethylene glycol and the interfacial tension that exists between the two polymer solutions.
JoVE Bioengineering
Synthetic, Multi-Layer, Self-Oscillating Vocal Fold Model Fabrication
Department of Mechanical Engineering, Brigham Young University
The methodology for fabricating synthetic vocal fold models is described. The models are life-sized and mimic the multi-layer structure of the human vocal folds. Results show the models to self-oscillate at pressures comparable to lung pressure and demonstrate flow-induced vibratory responses that are similar to those of human vocal folds.
JoVE General
Introduction to Solid Supported Membrane Based Electrophysiology
1Department of Biophysical Chemistry, Max Planck Institute of Biophysics, 2Buchmann Institute for Molecular Life Sciences, Goethe University Frankfurt
Here we present an electrophysiological method based on solid supported membranes with focus on its applications for the characterization of electrogenic membrane transporters.
JoVE General
A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example
In this article, a simple, quantitative, liquid phase affinity capture assay is presented. It is a reliable technique based on the interaction between magnetic beads and tagged proteins (e.g. nanobodies) on one hand and the affinity between the tagged protein and a second, labeled protein (e.g. poliovirus) on the other.
JoVE Neuroscience
Comprehensive Profiling of Dopamine Regulation in Substantia Nigra and Ventral Tegmental Area
Dopamine is distinctly regulated in the midbrain nuclei, which contain the cell bodies and dendrites of the dopamine neurons. Here we describe a dissection and sample-handling approach to maximize results, and thus conclusions and insights, on dopamine regulation in the midbrain nuclei of the substantia nigra (SN) and ventral tegmental area (VTA) in rodents.
JoVE General
An In Vitro Skin Irritation Test (SIT) using the EpiDerm Reconstructed Human Epidermal (RHE) Model
In this video, we demonstrate the EpiDerm Skin Irritation test (EpiDerm SIT) developed and validated for in vitro skin irritation testing of chemicals, including cosmetic and pharmaceutical ingredients.
JoVE General
High-Throughput Measurement and Classification of Organic P in Environmental Samples
School of Engineering, University of Vermont
This protocol describes a high-throughput method of enzymatic hydrolysis that utilizes a microplate reader to measure and classify soil phosphorus as P monoesters, P diesters and inorganic P. Up to 96 samples can be measured at one time in a standard laboratory.
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